JP2017178866A - Method for producing adrenomedullin lyophilized preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide adrenomedullin lyophilized preparations.SOLUTION: An adrenomedullin lyophilized preparation is produced by the following process consisting of the steps: (i) adrenomedullin or a modified body thereof is dissolved in an aqueous solution substantially free from buffer components, to prepare a peptide solution; and (ii) the peptide solution prepared in the step (i) is lyophilized.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a lyophilized preparation containing adrenomedullin or a modified product thereof as an active ingredient.

    Adrenomedullin (hereinafter also referred to as “AM”) is a physiologically active peptide isolated and identified from brown cell tissue in 1993 (Non-patent Document 1). Initially, AM was found to exert a potent vasodilatory antihypertensive effect. For example, Patent Document 1 describes a peptide having an antihypertensive action comprising the amino acid sequence of human AM.

  Subsequent research has revealed that AM is a multifunctional physiologically active substance that exhibits various pharmacological actions such as cardiovascular protective action, anti-inflammatory action, angiogenesis action and tissue repair promoting action. In addition, with the aim of applying the pharmacological action of AM to the treatment of diseases, studies on the administration of AM to patients with various diseases have been conducted. For example, the usefulness of AM as a therapeutic agent for inflammatory bowel disease, peripheral vascular disease or acute myocardial infarction is expected (Non-patent Document 2).

  Patent Document 2 contains adrenomedullin or a derivative thereof having an activity of suppressing non-bacterial inflammation, or a salt thereof having an activity of suppressing non-bacterial inflammation as an active ingredient A preventive or therapeutic agent for nonbacterial inflammatory bowel disease is described.

  Patent Document 3 is a method for preventing or treating inflammatory bowel disease in a patient who needs prevention or treatment of inflammatory bowel disease where it is difficult or insufficient to use a steroid preparation, immunosuppressive agent or biological preparation. And administering an effective amount of adrenomedullin, a modified form thereof having an activity to suppress inflammation, or a salt of the adrenomedullin or the modified form and having an activity to suppress inflammation to the patient. The method for preventing or treating is described.

  Patent Document 3 discloses that synthetic human AM is dissolved in a 3.75% mannitol solution and sealed in a vial and stored frozen at -30 ° C. However, if the lysate enclosed in the vial is only stored frozen, the quality stability is insufficient and it cannot be stored as a preparation for a long period of time. In particular, with respect to peptide preparations, it is difficult to obtain a quality-stable preparation because the preparation tends to be easily denatured such as degradation, aggregation and precipitation of peptides over time.

Japanese Patent No. 2774769 Japanese Patent No. 4830093 International Publication No. 2012/096411

Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun, April 30, 1993, Volume 192 (2) , pp. 553-601 Kato, J., Kitamura, K .. Bench-to-bedside pharmacology of adrenomedullin. European Journal of Pharmacology, 2015, 764, p. 140-148.

  In such cases, lyophilized preparations are usually examined. In the production of freeze-dried preparations, increasing the amount and concentration of the active substance and pharmaceutical additives will include stability and other preparation characteristics (eg appearance (color, shape, etc.), moisture content, etc.) There is a risk that quality important for pharmaceutical management may be impaired. For this reason, it is required to strictly design the solvent, blending amount, freeze-drying conditions, and the like. And the manufacturing method of AM freeze-dried preparation is not known until now.

  Therefore, an object of the present invention is to provide a method for producing an AM freeze-dried preparation.

  As a result of various studies on means for solving the above-mentioned problems, the present inventors have found an optimal solvent, blending amount, and freeze-drying conditions for an AM freeze-dried preparation. Based on the above findings, the present inventors have completed the present invention.

That is, the gist of the present invention is as follows.
(1) A method for producing a freeze-dried preparation comprising the following steps;
(I) A step of dissolving adrenomedullin or a modified form thereof in an aqueous solution substantially free of a buffer component to prepare a peptide solution; and (ii) a step of lyophilizing the peptide solution prepared in (i).
(2) The production method of (1), wherein the aqueous solution containing substantially no buffer component is an aqueous D-mannitol solution.

  According to the present invention, it is possible to provide an AM freeze-dried preparation excellent in long-term stability.

  In the present invention, adrenomedullin (AM) to be formulated is not only a human-derived peptide (SEQ ID NO: 1, Non-Patent Document 1) isolated and identified from human brown cell tissue, but also, for example, pig (SEQ ID NO: 4) Peptides (orthologs) derived from other non-human mammals (for example, warm-blooded animals) such as dogs (SEQ ID NO: 6), cows (SEQ ID NO: 8), rats (SEQ ID NO: 10) or mice (SEQ ID NO: 12) It may be. In vivo, in these peptides, two cysteine residues in the amino acid sequence form a disulfide bond, and the C-terminal is amidated. In the present specification, the peptide having a disulfide bond and a C-terminal amide group may be referred to as “natural adrenomedullin” or simply “adrenomedullin”. The present invention can be applied to any of the above peptides.

  As used herein, “C-terminal amidation” means one embodiment of post-translational modification of a peptide in vivo. Specifically, the main chain carboxyl group of the C-terminal amino acid residue of the peptide is an amide group. It means a reaction that is converted to a form. Further, in the present specification, “formation of disulfide bond of cysteine residue” or “disulfation of cysteine residue” means one embodiment of post-translational modification of a peptide in vivo. It means a reaction in which two cysteine residues in an amino acid sequence form a disulfide bond (-SS-). Many bioactive peptides produced in vivo are initially biosynthesized as precursor proteins with higher molecular weights, such as C-terminal amidation and / or disulfation of cysteine residues during the process of intracellular translocation. It undergoes a post-translational modification reaction and becomes a mature bioactive peptide. C-terminal amidation usually proceeds by the action of a C-terminal amidating enzyme on the precursor protein. In the case of a bioactive peptide having a C-terminal amide group, in the precursor protein, a Gly residue is bonded to the C-terminal carboxyl group to be amidated, and the Gly residue is C-terminal by a C-terminal amidating enzyme. Converted to an amide group. Further, the C-terminal propeptide of the precursor protein has a repeating sequence of a combination of basic amino acid residues such as Lys-Arg or Arg-Arg (Mizuno, Biochemistry Vol. 61, No. 12, 1435-1461 (1989)). Disulfidation of cysteine residues can proceed under oxidative conditions. In vivo, disulfation of cysteine residues usually proceeds by the action of protein disulfide isomerase on the precursor protein.

The adrenomedullin or a modified form thereof is as follows:
(I) a peptide comprising the amino acid sequence of adrenomedullin,
(Ii) a peptide consisting of an amino acid sequence of adrenomedullin, wherein two cysteine residues in the amino acid sequence form a disulfide bond,
(Iii) In the peptide of (ii), the disulfide bond is substituted with an ethylene group,
(Iv) A peptide in which 1 to 15 amino acids are deleted, substituted or added in any peptide of (i) to (iii),
(V) In the peptide of any one of (i) to (iv), the peptide in which the C-terminus is amidated, and in any of the peptides (vi) (i) to (iv), the glycine residue is present at the C-terminus. Peptides selected from the group consisting of peptides to which groups are added are preferred.

  In the peptides (i) to (vi), the amino acid sequence of adrenomedullin included in (v) is amidated at the C-terminus, and two cysteine residues in the amino acid sequence are disulfides. The peptide forming the bond corresponds to the mature natural adrenomedullin. The peptide consisting of the amino acid sequence of adrenomedullin in (i) corresponds to the natural adrenomedullin in the form before undergoing post-translational modification of C-terminal amidation and disulfation of cysteine residues (ie, immature). In the peptides (i) to (vi), the other peptides except the peptides described above correspond to adrenomedullin modifications.

  The peptide of (ii) is formed by air-oxidizing the thiol groups of the two cysteine residues of the peptide of (i) or oxidizing them with an appropriate oxidizing agent to convert them into disulfide bonds. Can be made. The three-dimensional structure of the peptide (ii) is substantially the same as the three-dimensional structure of natural adrenomedullin. Thereby, the activity of the modified form of adrenomedullin can be made substantially equivalent to that of natural adrenomedullin.

  The peptide of (iii) can be formed by converting the disulfide bond of the peptide of (ii) to an ethylene group. The substitution from a disulfide bond to an ethylene group can be performed by a method well known in the art (O. Keller et al., Helv. Chim. Acta, 1974, vol. 57, p. 1253). By using the peptide (iii), the three-dimensional structure of the peptide can be stabilized.

  In the peptide (iv), the number of amino acid residues that are deleted, substituted or added is preferably in the range of 1 to 12, more preferably in the range of 1 to 10, and 1 to 8 More preferably, the range is 1 to 5, particularly preferably 1 to 3, and most preferably 1 to 3.

  The peptide (vi) can be converted to the peptide (v) by converting the C-terminal glycine residue into a C-terminal amide group by the action of the C-terminal amidating enzyme. Therefore, by administering the peptide (vi) to a subject, a C-terminal amidated peptide can be formed in the subject's living body after a lapse of a certain time.

The adrenomedullin or a modified form thereof is as follows:
(A) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, or a peptide consisting of the amino acid sequence of SEQ ID NO: 1, wherein the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond;
(B) a peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4, wherein the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond;
(C) a peptide consisting of the amino acid sequence of SEQ ID NO: 6, or a peptide consisting of the amino acid sequence of SEQ ID NO: 6, wherein the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond;
(D) a peptide consisting of the amino acid sequence of SEQ ID NO: 8, or a peptide consisting of the amino acid sequence of SEQ ID NO: 8, wherein the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond;
(E) a peptide consisting of the amino acid sequence of SEQ ID NO: 10, or a peptide consisting of the amino acid sequence of SEQ ID NO: 10, wherein the cysteine residue at position 14 and the cysteine residue at position 19 form a disulfide bond;
(F) a peptide consisting of the amino acid sequence of SEQ ID NO: 12, or a peptide consisting of the amino acid sequence of SEQ ID NO: 12, wherein the cysteine residue at position 14 and the cysteine residue at position 19 form a disulfide bond;
(G) The peptide according to any one of (a) to (f), wherein the disulfide bond is substituted with an ethylene group;
(H) a peptide in which 1 to 15 amino acids are deleted, substituted or added in any of the peptides of (a) to (g);
(I) a peptide in which the C-terminus is amidated in any of the peptides (a) to (h); and (j) in any of the peptides (a) to (h), Peptides to which glycine residues have been added;
More preferably, the peptide is selected from the group consisting of:

  In the peptide of (h), the number of deleted, substituted or added amino acid residues is preferably in the range of 1 to 12, more preferably in the range of 1 to 10, and 1 to 8 More preferably, the range is 1 to 5, particularly preferably 1 to 3, and most preferably 1 to 3.

  The production method of the present invention includes a step of preparing a peptide solution by dissolving the AM or a modified product thereof in an aqueous solution substantially free of a buffer component.

  This is because it has been found that when a buffer component is included when preparing a peptide solution of AM, salting out occurs due to the polarity (chargeability) of AM. Here, the buffer component is a component having an effect of maintaining or controlling the pH of the preparation in a predetermined pH range, and typically a buffer such as phosphate, acetate, carbonate, citrate is added. means. “Substantially” means an inorganic acid such as hydrochloric acid and phosphoric acid and its salt, an organic acid such as acetic acid, citric acid and lactic acid and its salt, It means that hydroxides such as sodium oxide, potassium hydroxide and calcium hydroxide may be included. The final adjustment of the pH of the lysis solution is performed as necessary. That is, this step includes a “step of dissolving in an aqueous solution not containing a buffer component” and a subsequent step of “finally adjusting the pH of the solution, which is performed as necessary”.

  The pH of the finally obtained peptide solution is preferably in the range of 3.0 to 9.0, more preferably in the range of 4.0 to 7.0. The pH is not particularly limited, but sodium hydroxide or hydrochloric acid is preferably used.

  In the peptide solution, additives described in the Pharmaceutical Additives Encyclopedia (Pharmaceutical Daily) can be blended as necessary. Examples of additives include suspending agents (benzalkonium chloride, sodium lauryl sulfate, laurylaminopropionic acid, glyceryl monostearate, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, etc.), dispersing agents ( Sodium citrate, light aluminum oxide, polysorbate, macrogol, dextrin, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, etc.), emulsifier (lysine, propylene glycol, cetanol, lecithin, lanolin, sodium lauryl sulfate, etc.), surfactant (Cetanol, polyoxyethylene cetyl ether, lauromacrogol, etc.), isotonic agents (glucose, D-sorbitol, D-mannitol, glycerin) Phosphorus, sodium chloride, etc.), a soothing agent (creatinine, benzyl alcohol, etc.), stabilizing agents (taurine, amino acids, p-hydroxybenzoic acid esters, benzyl alcohol, crystalline cellulose, macrogol, etc.). These additives can be used alone or in appropriate combination.

  In the most preferred embodiment of the present invention, AM is dissolved in water for injection to which only D-mannitol as an isotonic agent is added, and the pH is finally adjusted with sodium hydroxide or hydrochloric acid as necessary to prepare a peptide solution. Is.

  The amount of D-mannitol to be added is 1 mg to 2500 mg of D-mannitol per 1 mg of AM, preferably 50 to 200 mg of D-mannitol, and more preferably 100 mg of D-mannitol. When a freeze-dried preparation is used as an injection after re-dissolution, an optimal osmotic pressure ratio can be obtained by setting such a blending amount.

  The peptide solution obtained as described above serves as a raw material for a preparation excellent in stability and other preparation characteristics by suppressing the amount to be lyophilized in the next step to the minimum necessary amount.

  The obtained peptide solution is subjected to sterilization filtration using a sterilization filter. As the sterilization filter, a sterile filter having a nominal pore size of 0.22 μm (or smaller) or a filter having at least the same ability to collect microorganisms is used. Thereafter, the peptide solution is injected into a vial or ampoule, and the next lyophilization step is performed.

  In this invention, the process of freeze-drying the said peptide solution is further included.

  “Freeze-drying” refers to a step of injecting a predetermined amount of the above peptide solution into a container and drying it by freeze-drying to obtain a solid. Specifically, a freezing process that cools and freezes under normal pressure, a primary drying (sublimation) process that sublimates and drys free water that is not constrained by the solute, and a secondary drying (desorption) that removes adsorbed water and crystal water inherent to the solute. There are three methods including a wet process. The temperature suitable for each process is -60 ° C to -40 ° C in the freezing process, -50 ° C to 0 ° C in the primary drying process, and 4 ° C to 40 ° C in the secondary drying process. The vacuum pressure in the primary drying process is preferably controlled at 0.1 to 50 Pa, more preferably 1 to 20 Pa. The vacuum pressure in the secondary drying process is preferably controlled at a higher vacuum. For example, it is preferably performed in a high vacuum state of 0.01 to 10 Pa.

  After finishing each of the above processes, the inside of the drying chamber is decompressed. As a method of restoring pressure, aseptic air or inert gas (for example, sterile nitrogen gas, sterile helium gas, etc.) is sent into the chamber to perform primary decompression to about 60 to 90 kPa, and then further if necessary. A method of restoring pressure (secondary restoring pressure) is preferable. The vial is preferably stoppered after the primary decompression.

  In the most preferred embodiment of the present invention, the freezing process is performed at −50 ° C., the primary drying process −50 ° C. · 10-15 Pa, the secondary drying process 30 ° C., and high vacuum (about several Pa or less).

  The AM freeze-dried preparation obtained by the production method of the present invention is used after redissolving with an injection solution. Re-dissolution can be performed by a normal method.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

1. Production of lyophilized preparations
(1) Peptide drug substance synthetic human adrenomedullin (a peptide consisting of the amino acid sequence of SEQ ID NO: 1, Cys at position 16 and Cys at position 21 are disulfide-bonded, and the C-terminal is amidated) The product synthesized according to the same standards (Good Manufacturing Practice: GMP) as pharmaceutical manufacturing was purchased as a drug substance. The purity was 99% or more.

(2) Preparation of peptide solution
(a) About 6000 g of water for injection was added to 10 L Nalgen, 79.5 g of D-mannitol (Mitsubishi Food Tech) was added, washed with water for injection, and stirred and dissolved at room temperature.
(b) After confirmation of dissolution, 826.8 mg of human adrenomedullin (corrected by the purity and peptide content of the drug substance) was added, and after washing with water for injection, the mixture was stirred and dissolved at room temperature.
(C) After confirming dissolution, the amount was temporarily increased to 7300 g with water for injection and further stirred at room temperature.
(d) If the pH of the above solution is not in the range of 4.0 to 7.0, adjust the pH with a 1 mol / L sodium hydroxide solution or a 1 mol / L hydrochloric acid solution as necessary.
Thus, a peptide solution is obtained.

(3) Aseptic filtration (e) It filtered with the filter for aseptic filtration (0.22 micrometer).

(4) Lyophilization (f) The filtered peptide solution is filled in a vial (Fuji Glass) and freeze-dried by a freeze-dryer (Kyowa Vacuum Technology).
(F-1) Freezing process: The solution was gradually cooled from room temperature to −50 ° C. and kept at −50 ° C. for 4 hours.
(F-2) Primary drying process: 13.3 Pa It carried out under pressure reduction control. The temperature was raised to −10 ° C. and held for 40 hours or more.
(F-3) Secondary drying process: It was carried out under reduced pressure control of the maximum vacuum (1.3 Pa or less) of the apparatus. The temperature was raised to 30 ° C. and held for 6 hours or more.
(G) After lyophilization, the pressure was restored with nitrogen, and a slight negative pressure stopper was applied at -0.016 MPa.
By the above procedure, a freeze-dried preparation of adrenomedullin 0.5 mg and D-mannitol 50 mg / 1 vial (5 mL) could be obtained.

  The obtained freeze-dried preparation was tested in accordance with the Japanese Pharmacopoeia General Test Method Moisture Measurement Method (Karl Fischer Method) and coulometric titration, and confirmed that the moisture content was 5.0% or less.

2. Quality Confirmation The freeze-dried preparation obtained as described above was placed under accelerated conditions (temperature: 25 ± 2 ° C., humidity: 60 ± 5%), and the change with time in quality over two months was evaluated.

  (1) A test sample was prepared by adding 16 mL of physiological saline to one vial of the lyophilized preparation prepared through the steps (a) to (g). As a standard sample, a peptide solution prepared through the steps (a) to (d) is used. The retention time and mass spectrum were confirmed by a high performance liquid chromatograph / mass spectrometer (LCMS-8030, Shimadzu Corporation) according to the Japanese Pharmacopoeia General Test Method Liquid Chromatography and Mass Spectrometry.

  As shown in Table 1, the elution time of the freeze-dried preparation and the standard sample showed no difference between the two samples.

  As shown in Table 2, the detection mass spectra of the lyophilized preparation and the standard sample showed no difference between the two samples.

(2) A test sample was prepared by adding 5 mL of physiological saline to one vial of the lyophilized preparation prepared through the steps (a) to (g). Japan Pharmacopoeia General Test Method Purity (methionine oxidant) was confirmed by high performance liquid chromatograph / mass spectrometer (LCMS-8030, Shimadzu Corporation) according to liquid chromatography. The amount of oxidized methionine was calculated from the peak area of oxidized methionine obtained from the test sample and the peak area of adrenomedullin obtained from the standard solution.

  As shown in Table 3, it was confirmed that the amount of oxidized methionine showed a low value and maintained high quality.

(3) A test sample was prepared by adding 5 mL of physiological saline to one vial of the lyophilized preparation prepared through the steps (a) to (g). Japanese Pharmacopoeia General Test Method Purity (related substances) was confirmed by high performance liquid chromatograph / mass spectrometer (LCMS-8030, Shimadzu Corporation) according to liquid chromatography. The amount of the related substance was calculated by the area percentage method by measuring the peak area of each test sample by the automatic integration method.

  As shown in Table 4, it was confirmed that the maximum amount of related substances and the total amount of related substances showed low values and maintained high quality.

(4) In addition, the quality of each of the osmotic pressure ratio, pH, moisture, formulation uniformity, insoluble foreign matter, insoluble microparticles, endotoxin, sterility, and quantitative properties conforms to the test methods specified by the Japanese Pharmacopoeia General Test Method. Confirmed. As a result, it was confirmed that in all tests, high quality was maintained even after 2 months under accelerated conditions.

Claims (2)

A method for producing a freeze-dried preparation comprising the following steps;
(I) A step of dissolving adrenomedullin or a modified form thereof in an aqueous solution substantially free of a buffer component to prepare a peptide solution; and (ii) a step of lyophilizing the peptide solution prepared in (i). The manufacturing method of Claim 1 whose aqueous solution which does not contain a buffer component substantially is D-mannitol aqueous solution.